A basic premise in most studies of mitochondrial oxidative phosphorylation has been that energy from electron transport serves in some manner to drive the formation of the terminal covalent anhydride bond in ATP. Recent results which we have obtained have led us to propose a distinctly different mode of energy input for net ATP synthesis: namely, that energy is used to bring about the release of preformed, tightly-bound ATP. Our working hypothesis predicts that a major energy-requiring step is the decrease in binding affinity for ATP at the catalytic site. We propose to test this prediction by performing equilibrium binding measurements using gel exclusion and filtration methods. A nonhydrolyzible imido analog of ATP will be incubated with submitochondrial particles under different metabolic conditions. The effects of inhibitor protein, aurovertin, and efrapeptin will be tested. Recent evidence suggests that energy from oxidation-reduction reactions of the respiratory chain may also serve to promote binding of ADP and Pi. The energy-dependence of ADP binding will be determined under conditions described for the ATP analog. The energy dependence of Pi binding will be assessed by measurement of the Km of Pi and the Ki of Asi, for the Pi reversibly yields HOH exchange. A second prediction of our hypothesis is the reversible formation of ATP plus HOH from ADP plus Pi at the catalytic site, accompanied by a Pi reversibly yields HOH exchange reaction, even though sufficient energy for net synthesis of medium ATP is not available. As a critical test of this prediction, we propose to assay for these activities using isolated coupling factors which catalyze an oligomycin-sensitive ATPase and Pi reversibly yields ATP exchange. An additional objective of this proposal is to determine the structural relationship of the mitochondrial adenine nucleotide translocase to the coupling factors of oxidative phosphorylation. The effect of carboxyatractyloside, a competitive inhibitor of the translocase, will be tested on adenine nucleotide binding to isolated coupling factors.